PROJECT SUMMARY The overarching goal of this proposal is to further our understanding of evolutionarily conserved mechanisms that modulate mitochondrial proliferation and energy metabolism. Although significant progress has been made in understanding the consequences of mitochondrial dysfunction and characterizing the emergent disease, it remains unclear whether manipulation of overall mitochondrial proliferation to alter mitochondrial number and/or mass would impact on disease phenotypes. This conceptual gap exists largely due to the genetic redundancy, relative intractability, and functional complexity associated with regulation of mitochondrial proliferation and function in mammals. To mitigate these limitations, mitochondria are often studied in Drosophila, which are genetically tractable organisms characterized by extensive conservation with mammals in terms of mitochondrial biology. However, transcriptional networks that regulate mitochondrial proliferation and function are unknown in Drosophila. We have now identified a master transcriptional regulator of mitochondrial proliferation in Drosophila that is both necessary and sufficient to determine mitochondrial mass. Thus, we are in a position to finally delineate the relationship between mitochondrial proliferation and function. In Aim 1, we will test the hypothesis that the transcription factor we have identified modulates mitochondrial function in addition to mitochondrial mass. Successful completion of this aim could present unprecedented opportunities for en masse modulation of mitochondrial function in Drosophila models of human diseases. In Aim 2, we will examine the interrelationship between AMPK, a known regulator of mitochondrial biogenesis and metabolism, with the identified regulatory network. These studies could reveal previously unrecognized, fundamental mechanisms by which AMPK regulates mitochondrial function. In Aim 3, we will test whether the signaling network and the human homologs of the identified transcription factor regulate mitochondrial biogenesis and/or function in the human cell. Taken together, our experimental strategies are designed to reveal novel conceptual insights into the regulation of mitochondrial proliferation and function. Furthermore, our multidisciplinary and systems-based approach will enable a deeper understanding of the pathophysiology of mitochondrial diseases. We hope that upon completion of these studies, we and other biomedical researchers can leverage the insights gleaned to inform innovative avenues for therapeutic intervention for treating human diseases involving mitochondrial dysfunction.